Structures and Anti-Inflammatory Evaluation of Phenylpropanoid Derivatives from the Aerial Parts of Dioscorea polystachya

Seven undescribed phenylpropanoid constituents, including three new bibenzyl derivatives (1–3) along with four new benzofuran stilbene derivatives (4–7), were isolated from the aerial parts of Dioscorea polystachya. The structures of these compounds were elucidated using a combination of spectroscopic analyses, including UV, IR, HRESIMS, 1D, and 2D NMR. Further, all the compounds were evaluated on the anti-inflammatory activity for their inhibition of nitric oxide (NO) production by RAW 264.7 macrophages cells, and some of them (1–3 and 6) displayed inhibitory activity with IC50 values in the range of 9.3–32.3 μM. Moreover, compound 3 decreased the expression of iNOS in Western blot analysis, suggesting compound 3 is mediated via the suppression of an LPS-induced NF-κB inflammasome pathway.


Introduction
The genus of Dioscorea is a perennial herbaceous and monocotyledon plant, and has large roots and/or rhizomes [1][2][3][4]. Dioscorea plants are widely distributed in China. The D. polystachya tuber has been edible with a history of more than 3000 years in China, and it is also an important traditional Chinese herbal plant. It is used for the treatment of atherosclerosis, coughing with lung heat, hypertension, pyretic stranguria, anthracia, hyperglycemia, coronary heart disease, swelling, gastric ulcers, and sprains [5][6][7][8][9]. Previous phytochemistry investigations focused on the genus of Dioscorea and resulted in the isolation of bibenzyls, phenanthrenes, biphenanthrenes, lignans, steroidal saponins, flavonoids, and polysaccharides [10][11][12].
However, there is still a deficiency of bioactive compounds from the aerial parts D. polystachya. With our ongoing research of chemical constituents and biological activity with herbal medicine [13][14][15], phytochemical studies of the aerial parts of D. polystachya were carried out in this research in consideration of explore anti-inflammatory ingredients. Here, seven undescribed phenylpropanoid constituents, including three new bibenzyl compounds (1)(2)(3) along with four new benzofuran stilbene compounds (4)(5)(6)(7), were isolated from the aerial parts of D. polystachya ( Figure 1) and their chemical structures were elucidated based on UV, IR, NMR, and HRESIMS spectroscopic data. Compound 3, a bibenzyl compound, exhibits strong anti-inflammatory effect, and it should inhibit the inflammatory signaling mechanism of NF-κB pathways. In this report, their isolation, structural characterization, and potential anti-inflammatory activities are described.

Isolation of New Compounds 1-7 from the Aerial Parts of D. polystachya
The fresh aerial parts of D. polystachya (5 kg) were extracted three times with methanol to give a crude extract, then the extract was suspended in water and extracted with EtOAc, which were subjected to MCI, C 18 column chromatography, MPLC, preparative HPLC, and preparative TLC to obtain seven new phenylpropanoid compounds 1-7 ( Figure 1).
Compound 2 was obtained as a yellowish solid powder. Its molecular formula had the same HRESIMS data as compound 1, indicating they were isomers. The NMR data ( Figure S2) showed that compounds 1 and 2 had similar structures. Nevertheless, they have different polars in HPLC analysis. The 1D NMR spectra (Table 1)  In the HMBC spectrum (Figure 2), the correlations from H-2/6 to C-4 revealed that the methoxy group was linked to C-4. The HMBC correlations from H-2 to C-3 (δ C 151.2) indicated that a hydroxy group was linked to C-3. Therefore, compound 2 was determined as 4,5,2 -trimethoxy-3,3 -dihydroxy-bibenzyl and named diosbiben B.  Compound 3, a faint yellow solid, had the molecular formula of C 17 H 20 O 5 from HRESIMS data and an unsaturation of 8. The UV spectrum of 3 was similar to 1 and 2. Typical absorption peaks attributed to broad hydroxyl (3290 cm −1 ), aromatic ring (1612, 1518 cm −1 ), and methine (1474 cm −1 ) functionalities were observed in the IR spectrum. Its molecular formula and NMR data ( Figure S3) were similar to that of compounds 1 and 2, indicating that they were possible isomers and also were bibenzyl compounds. The HMBC correlations from H-2/6 to C-4 (δ C 133.1) indicated a hydroxy group was linked to C-4. One methoxy group was linked to C-3 , which was also demonstrated by the following correlations in the HMBC spectrum: H-5 to C-3 (δ C 154.0); 3 -OCH 3 to C-3 . Finally, the structure of 3 was identified as 5,2 ,3 -trimethoxy-3,4-dihydroxy-bibenzyl and named diosbiben C.

Inhibitory Effects of New Compounds 1-7 on NO Production of LPS-Activated RAW 264.7 Cells
All the isolates were tested for their effects on nitric oxide (NO) production inhibition in lipopolysaccharide (LPS)-activated RAW 264.7 cells. In order to exclude the inhibition of NO production caused by cytotoxicity, cell viability was evaluated by the MTT method. Results revealed that no obvious cytotoxicity (over 75% cell survival) for most of compounds at concentrations up to 50 µM was observed. The results shown in Table 4, most of the isolated compounds (1-3, 6) that displayed NO inhibitory activity (positive control: Aminoguanidine hydrochloride, IC 50 19.2 ± 0.78 µM). The NO inhibitory activity of compounds 1-3 (IC 50 9.3-32.3 µM) versus 6 (IC 50 24.1 µM) suggested the bibenzyl compounds were more active than benzofuran stilbene compounds. The position of substituent may play a significant role in mediating the activities.

Inhibitory Effects of New Compound 3 on LPS-Enhanced Inflammatory Mediators
Since compound 3 displayed the strongest inhibition on NO produce of all isolated compounds, so it was chosen for mechanistic research. Compound 3 displayed no cytotoxicity at concentrations up to 25 µM ( Figure 4A); thus, this concentration was used in subsequent experiments. As shown in Figure 4B, the significant NO production inhibitory effect of 3 with an IC 50 of 9.3 ± 1.03 µM was observed. As is well known, the production of NO is closely related to the key proteins iNOS and COX-2 [18]. Therefore, the expression levels on these proteins were detected by Western blotting. As shown in Figure 4C, a mild down-regulation of COX-2 expression was observed, but the levels of iNOS were inhibited in the presence of 3 at 10 µM. Based on these observations, it was inferred that 3 exhibited inhibitory effects on NO production may by means of suppression of iNOS in LPS-induced RAW 264.7 macrophages.

Discussion
The biological activities of D. polystachya, which have not been fully demonstrated. Moreover, few studies have been conducted with the metabolites of the aerial parts of D. polystachya. In the current study, the secondary metabolites of the aerial parts of D. polystachya were investigated by chromatographic purification and interpretation of spectroscopic data to generate seven new phenylpropanoid derivatives, and their antiinflammatory activities were explored on LPS-induced inflammatory molecules in RAW 264.7 cells.
The NO is a key signaling molecule and has been well known to regulate various physiological functions in many tissues of the human body [19]. However, an overproduction of NO is associated with many inflammatory diseases, so discovery of natural bioactive compounds plays a key role in research of new drugs for reducing the inflammatory molecules. Therefore, seven new phenylpropanoid compounds were isolated from the methanol extract of the aerial parts of D. polystachya by many kinds of chromatographs. The inhibitory rate on LPS-induced expression of nitric oxide in RAW 264.7 cells of all isolated compound were evaluated, and the results showed that compounds (1-3, 6) that displayed NO inhibitory activity. Notably, by comparing compound 1 with 3, we found when the methoxy group located at C-3 could cause a dramatic reduction in the inhibitory activity. The NO inhibitory activity of compounds 1-3 versus 4-7 suggested the bibenzyl compounds were more active than benzofuran stilbene compounds. The anti-inflammatory activities of bibenzyl derivatives may due to their special chemical structure, in which a carbon-carbon bond can have free rotation that produces multiple spatial conformations. These results demonstrated that structurally different phenylpropanoid compounds in D. polystachya were possible to play their anti-inflammatory function. Furthermore, proinflammatory molecules, including iNOS and COX-2, were involved in inflammation-associated diseases and act as inflammatory mediators or activators of inflammatory pathways, such as NF-κB [20]. Therefore, the expression levels on these proteins were detected by Western blotting. The result show that compound 3 obvious inhibited the expression of iNOS, which proposing compound 3 appears to be mediated NO release via the suppression of NF-κB inflammasome pathway.
In this study, a bibenzyl derivative, diosbiben C (3), isolated from the aerial parts of D. polystachya, significantly reduced the NO release of LPS-stimulated in RAW 264.7 cells, and the Western blot analysis resulting in a reduction in the expression inflammatory molecules of iNOS in NF-κB pathway. In addition to the NF-κB signaling pathway, our future research will investigate the anti-inflammatory mechanism of diosbiben C in more detail.

Plant Materials
The fresh aerial parts of D. polystachya were collected at Lu'an, Anhui province (China), in September 2019 and identified by associate Prof. Tao Xu.

Extraction and Isolation
The fresh aerial parts of D. polystachya (5 kg) were extracted three times with methanol to give a crude extract, then the extract was suspended in water and extracted with EtOAc, affording an EtOAc soluble extract (20 g). The extract was subjected to an MCI gel column CC (46-50 µm) using MeOH-H 2 O (v/v) in step gradient from 1:9 to 1:0 to obtain four fractions Fr.1-Fr.4. Fr. 2 (800 mg) was subjected to Sephadex LH-20 column (MeOH) and separated by semipreparative HPLC (MeCN-H

Cell Viability Assay
An MTT assay was used to evaluate RAW 264.7 cell viability as previously described. Briefly, cells were plated in 96-well plates (5 × 10 3 cells/well) for 18 h and then incubated with compounds 1-7 in various concentrations with or without LPS (1.0 µg/mL). Eighteen hours later, the prepared MTT solution (20 µL, 5 mg/mL) was added, and the cells were incubated for another 4 h. After the formazan that formed was fully dissolved in DMSO (150 µL/well), the absorbance was read at 570 nm on a microplate reader. The viability of RAW 264.7 cells for the control group (with DMSO only) is defined as 100%.

Cell Culture and NO Production Measurements
The experimental procedures were followed by the literature [21]. Cells were seeded in 96-well plates at the density of 50,000 cells/well for 24 h, pretreated with the tested compounds for 30 min at 37 • C, and co-incubated with LPS (100 ng/mL) for 24 h. NO production was analyzed through Griess reaction. Momently, cell culture supernatant (50 µL) and Griess reagent (50 µL) were mixed for 10 min, and then monitored at 540 nm using a microplate reader. All the tested compounds were prepared as stock solutions with a concentration of 10 mM in DMSO. Aminoguanidine hydrochloride was used as the positive control.

Western Blot Analysis
Cells were pretreated with the test compound 3 for 30 min and stimulated with LPS (1 µg/mL) for 24 h. The total proteins were extracted and immunoblotted as previously described [22,23]. Briefly, the harvested cells were lysed by 1% RIPA (radioimmunoprecipitation assay) (Amresco, Solon, OH, USA) to achieve the cellular lysates. Cellular lysates were centrifuged, and the total protein concentration was measured by the BCA protein assay. Total proteins were electrophoresed on SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were washed with TBST buffer, blocking with 5% skim milk for 2 h at 25 • C, and then incubated with primary antibodies for 12 h at 4 • C. After being washed with TBST buffer, the membranes were treated with secondary antibody at room temperature.

Conclusions
In summary, seven undescribed phenylpropanoid derivatives (1-7) were isolated from the fresh aerial parts of D. polystachya. The new compounds were identified as bibenzyl and benzofuran stilbene derivatives. Those compounds will enrich the structural skeletons of natural occurring phenylpropanoids and the structural diversity of the Dioscoreaceae family. All new phenylpropanoid derivatives were screened for anti-inflammatory effects. The screened results revealed that compounds (1-3, 6) displayed strong NO inhibitory activity with the IC 50 range of 9.3-32.3 µM. Importantly, the potential compound 3 decreased iNOS levels, and indicated that 3 may mediated via the suppression of an LPS-induced NF-κB inflammasome pathway. These findings provide an insight into the potential therapeutic value of phenylpropanoid derivatives for inflammatory diseases. However, more studies are needed to determine whether these compounds can act on other inflammatory mechanisms.